Detection of the presence or absence of particular nucleic acid sequences in samples by use of nucleic acid amplification techniques has proven a powerful diagnostic tool with a multitude of applications.
The introduction of the polymerase chain reaction (PCR) provided the possibility for a rapid and specific amplification of specific target nucleic acid sequences present in samples. The PCR technique thereby provided a significant diagnostic tool for the detection of nucleic acid sequences in samples, and the introduction of this technique opened a new field of diagnostic applications.
Today, the polymerase chain reaction is well known to the skilled person.
However, it has proven to be difficult to detect the presence or absence of multiplex target nucleic acid sequences present in low copy numbers in a sample. Detecting several target sequences by parallel simplex PCR, each with a single primer set specific for the pathogen, are often not feasible due to a limited quantity of target DNA in the starting material. Alternatively, multiplex PCR using multiplex primer sets in the same reaction may be limited in detection sensitivity due to the inherent variability of amplification efficiencies of the different primer sets. Further, the PCR technique has proven to be time-consuming and difficult to handle in practice in a number of applications due to the requirement of a multitude of repeating cycles of thermal cycling (cycles of temperature shifts where different steps in the PCR process are taking place). The requirement of thermal cycling also causes problems in the manufacture of suitable detection devices where the requirements for rapid cooling and heating result in technical design problems for the device manufacturer. In order to provide a simpler device, an isothermal amplification technique would be more suitable.
To circumvent this and other difficulties, a number of alternative methods for the amplification of nucleic acid sequences have been developed, e.g. isothermal techniques such as transcription mediated amplification (TMA) or self-sustained sequence replication (3SR), nucleic acid sequence-based amplification (NASBA), signal-mediated amplification of RNA technology (SMART), strand displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification of DNA (LAMP), isothermal multiple displacement amplification (IMDA), helicase-dependent amplification (HDA), single primer isothermal amplification (SPIA), and circular helicase-dependent amplification (cHDA).
In a number of applications, however, only a very low copy number of target nucleic acid sequences are present in the sample under investigation. The presence of only very low copy numbers of nucleic acid sequences in a sample provides a challenge to any existing nucleic acid amplification technique.
As an example, early detection of sepsis may become a key factor for the successful diagnosis and treatment of patient suffering from that disease. However, only very minute amounts of microbial cells in the blood of e.g. humans may be present even in life threatening conditions of sepsis. Thus, in particular the detection of target nucleic acid sequences originating from microbial organisms causing sepsis poses great challenges to molecular diagnostic tools.
Similarly, the detection of different markers in HIV-infected patients poses significant difficulties in detection and diagnosis.
In a number of diagnostic applications it is critical to provide a diagnostic result within the shortest possible time. For example, due to the very rapid progression of sepsis, a key factor in a successful diagnosis of sepsis is the speed at which a diagnosis may be finalised.
In the diagnosis of sepsis, the extremely low number of bacteria in the blood and the corresponding extremely low number of specific nucleic acid marker sequences often present a significant challenge to the nucleic acid detection technique used. Further, the need of simultaneous investigation of the presence of multiple target nucleic acid sequences, e.g. in the diagnosis of sepsis, provides a further challenge in that the samples must be subject to a technique capable of amplifying and detecting multiple mutually different target nucleic acid sequences. Often, there is a need for simultaneous amplification of 10 or more sequences.
Loop-mediated amplification has proven to be a rapid, sensitive and highly specific technique for amplification of nucleic acid target sequences. However, even loop-mediated nucleic acid amplification has proven to be incapable in a number of applications, i.e. with an unsatisfactory sensitivity. Further, loop-mediated amplification cannot be used for the simultaneous detection of more than one target nucleic acid in a particular sample.
Nested PCR is a well-known technique, which provides a method of detecting very small amounts of target nucleic acid molecules in samples. The technique was introduced in order to circumvent the lack of sufficient sensitivity of conventional PCR. Nested PCR consists of a first step of PCR amplification of the target nucleic acid in the sample using a pair of primers targeted at the amplification of a nucleic acid sequence comprising the target sequence and a sequence upstream and downstream of the target, followed by a second step of PCR amplification of the target sequence. Thereby, a highly sensitive and specific detection of the target nucleic acid is achieved. However, nested PCR has proven to be time-consuming and practically difficult to handle in a number of applications. For example, multiplex nested PCR has shown to be difficult e.g. due to the multiple primer pairs required.
Existing techniques of multiplex detection of nucleic acid sequences, e.g. multiplex PCR, have in practice been difficult to operate with more than 3 or 4 target sequences. Further, multiplex PCR suffers to an even greater extent from the above described problems for PCR in general. Further, multiplex detection often requires that every target sequence is present in significant and approximately equal amounts in order to avoid an uneven amplification of the different target sequences. Assay approaches that split the initial specimen for parallel PCR reactions are often not feasible due to limited quantity of target DNA. Multiplex PCR using multiple primer sets in the same reaction may decrease specimen consumption but is limited in detection sensitivity.
Hence, the detection of several target sequences in e.g. the diagnosis of sepsis using multiplex PCR has turned out to be problematic.
Further, PCR is desirably to be avoided in a method suitable for use in a point-of-care system. This is primarily due to the requirement of temperature cycling, which may be problematic in a multiplex setting. Most of all, temperature cycling is problematic in the detection part or the detection sequence of steps in the method because measurement artefacts may be introduced by the presence of bubbles and/or the entire sample may be difficult to focus.
Accordingly, a method suitable for use in a point-of-care test, which method can simultaneously detect multiple pathogens from a single specimen, and which method does not use PCR in the final amplification steps is highly desirable.
Accordingly, there is a need in the art for more sensitive molecular techniques for the detection of target nucleic acid sequences in a sample.
There is also a need in the art for less time-consuming molecular techniques for the detection of target nucleic acid sequences in a sample.
There is also a need in the art for molecular techniques capable of simultaneous detection of two or more mutually different target nucleic acid sequences in a sample.
In particular, there is a need in the art for molecular techniques that are more sensitive, less time-consuming and allow for the detection of two or more mutually different target nucleic acid sequences in a sample.
There is also a need in the art for new molecular techniques that are more easily subjected to automated operation, i.e. requiring a minimal amount of time and where final amplification and detection can be performed under isothermal conditions in the same operation and within the same reaction vessel.
In the art of the diagnosis of bacteria related to sepsis, the above mentioned needs are especially pronounced.
It is therefore an object of the present invention to provide a simple and highly sensitive method of detecting the presence or absence of a target nucleic acid sequences in a sample.
It is a further object of the invention to provide a rapid method of detecting the presence or absence of a target nucleic acid sequences in a sample.
It is a further object of the present invention to provide a method of detecting the presence or absence of two or more mutually different target nucleic acid sequences in a sample.
It is a further object of the present invention to provide a method of detecting the presence or absence of two or more mutually different target nucleic acid sequences in a sample, the method being capable of detecting the presence or absence of very small amounts of target molecules in the sample.
It is further an object to provide a method for the amplification of two or more target molecule sequences related to the diagnosis of sepsis.
It is further an object of the invention to provide an amplification technique that is more easily subjected to automated operation, i.e. requiring a minimal amount of time and where final amplification and detection may be performed under isothermal conditions in the same operation and within the same reaction vessel.